A Glimpse at Siderophores Production by Anabaena flos-aquae UTEX 1444

In this study, a strain of Anabaena flos-aquae UTEX 1444 was cultivated in six different concentrations of iron (III). Cultures were extracted with organic solvents and analyzed using our dereplication strategy, based on the combined use of high-resolution tandem mass spectrometry and molecular networking. The analysis showed the presence of the siderophores’ family, named synechobactins, only in the zero iron (III) treatment culture. Seven unknown synechobactin variants were present in the extract, and their structures have been determined by a careful HRMS/MS analysis. This study unveils the capability of Anabaena flos-aquae UTEX 1444 to produce a large array of siderophores and may be a suitable model organism for a sustainable scale-up exploitation of such bioactive molecules, for the bioremediation of contaminated ecosystems, as well as in drug discovery.

Introduction
Living organisms require several kinds of metals for their wellness. In virtue of its unique coordination and redox chemistry, iron is, among the metal ions, involved in vital metabolic functions in plants, animals, and microorganisms. The two most frequent oxidation states of iron are +2 and +3, which are also referred to as ferrous and ferric, respectively. Because of its oxidation states, iron plays crucial roles in biocatalysis and electron transport chains, in several biological systems [1]. Microbes use different methods to collect iron from the environment; among others, they produce Fe 3+ -chelating molecules, named siderophores [2]. Siderophores are low-molecular-weight molecules (500-1500 Da), endowed with and named after their high affinity for iron (III) (Kf > 1030). Pathogens adapt their own iron-uptake strategy, in response to the type of infection (acute or chronic) and to the availability of iron [3] of the host. Acting as ferric ion scavengers, siderophores contribute significantly to the virulence of pathogenic microbes. Fe 3+ is transported inside the cell as a hexadentate octahedral complex with the siderophores. Depending on the primary oxygen-donating ligands that bind the iron, siderophores are classified as follows: hydroxamates, catecholates, carboxylates, and mixed-types siderophores [4].
Siderophores are powerful molecules that have applications in ecological research; for the removal of petroleum hydrocarbons from oceans and as algal bloom biocontrollers, in agriculture as soil bioremediators, and in drug discovery as iron chelation therapy, antibiotic carriers, and fish pathogen biocontrollers [5][6][7]. The most intriguing application is their use as ligands for antibiotics, the so-called "Trojan horse" strategy [8]. The potential Mar. Drugs 2022, 20, 256 2 of 10 chemical space of siderophore-antibiotic conjugates is a great opportunity to explore an untapped natural resource and design a new class of molecules to face drug-resistant pathogens [6,9].
Cyanobacteria are a class of photosynthetic microorganism, spread out in a large array of environments, from tropical areas to extremely cold waters. They are present in marine waters, as well as in freshwaters. This unique class of microorganisms is well known to produce several different classes of secondary metabolites, either toxins, named cyanotoxins, or bioactive natural products with interesting pharmacological properties [10]. Cyanobacteria rely on ferric iron to survive. While iron is one of the most abundant elements on Earth, bioavailable iron in freshwater and marine environments is limited, falling in the picomolar to a low nanomolar range. To survive in such an iron-depleted environment, cyanobacteria produce siderophores. Recently, it has been further speculated that cyanobacteria use siderophores as antimicrobial agents and as shields, to protect themselves from heavy-metal toxicity [11]. Cyanobacterial siderophores ( Figure 1) include mainly either hydroxamates or catecholates [12]. The first evidence of presence and types of siderophores in cyanobacteria date back to the early 1980s, when iron uptake in Anabaena sp. in iron starvation conditions was demonstrated to be mediated by the hydroxamate-type siderophore schizokinen [13]. On the other hand, cyanobacterial genomes are known to harbor a rich variety of gene clusters with unknown function. Recently, we reported on the awakening of a widely distributed class of silent gene clusters by iron starvation that yielded cyanochelin's production, β-hydroxy aspartate lipopeptides involved in iron uptake [14].
It is well known that a single strain produces different molecules when grown under different environmental conditions. This concept is the basis of the OSMAC ("One Strain, Many Compounds") strategy [15], a cultivation-based approach that consists in altering cultivation parameters, such as nutrient content, metal ions, rate of aeration, temperature, Mar. Drugs 2022, 20, 256 3 of 10 in order to trigger the productions of compounds of biomedical interest and to activate silent biosynthetic gene clusters.
In this context, in the frame of our ongoing research on the potentiality of cyanobacteria as incubators of bioactive molecules, we investigated the metabolome of the strain of Anabaena flos-aquae (UTEX 1444), when cultivated in the condition of iron deficiency and iron overload. Our study revealed the presence of a suite of synechobactins (seven of which are new variants), only in the extracts from the "zero-Iron (III) treatment" culture.
In this context, in the frame of our ongoing research on the potentiality of cyanobacteria as incubators of bioactive molecules, we investigated the metabolome of the strain of Anabaena flos-aquae (UTEX 1444), when cultivated in the condition of iron deficiency and iron overload. Our study revealed the presence of a suite of synechobactins (seven of which are new variants), only in the extracts from the "zero-Iron (III) treatment" culture.

Molecular Networking and Synechobactins' Identification
All extracts from cultures #1-6 were analyzed by LC-HRMS using an LTQ Orbitrap instrument. Data-dependent acquisition was used to trigger MS 2 scans of the ten most intense ions detected in the full MS scan. The raw LC-MS data were pre-processed using the MZmine program 2.53 [19], which allowed us to remove isotopic peaks, to identify adducts and to perform quantitation. The preliminary analysis (data not shown) of MZmine data revealed that the most significant extract for each culture was that of butanol, as the most representative of each entire metabolome. Therefore, the subsequent data processing and molecular networking were performed on the butanol extracts,

Molecular Networking and Synechobactins' Identification
All extracts from cultures #1-6 were analyzed by LC-HRMS using an LTQ Orbitrap instrument. Data-dependent acquisition was used to trigger MS 2 scans of the ten most intense ions detected in the full MS scan. The raw LC-MS data were pre-processed using the MZmine program 2.53 [19], which allowed us to remove isotopic peaks, to identify adducts and to perform quantitation. The preliminary analysis (data not shown) of MZmine data revealed that the most significant extract for each culture was that of butanol, as the most representative of each entire metabolome. Therefore, the subsequent data processing and molecular networking were performed on the butanol extracts, originating from #1-6 cultures. The global .mgf file, containing the MS 2 data from #1-6 butanol extract and the quantitation table obtained by mzMine were submitted to the online platform at the Global Natural Products Social Molecular Networking website [20], where a Feature-Based Molecular Network (FBMN) was generated https://gnps.ucsd.edu/ProteoSAFe/status. jsp?task=664d019a6b52486d880014bb1e16bd69 (accessed on 12 November 2021) [21]. The resulting molecular network was then visualized using the Cytoscape program 3.9.0 [22] (see also Figures S1-S13, Supplementary Material).
The comprehensive quali-quantitative network of #1-6 butanol extracts (Figure 3a) contains 165 features, grouped into 10 clusters. In the network, each node is represented as a pie chart showing the amount of the compound in the source cultures containing different iron concentrations (Figure 3b). One of the ten clusters was composed of 23 nodes, 13 of which were derived solely from the zero iron (III) culture (Figure 3c, nodes in red). These nodes did not match any known compounds in GNPS' library. The ESI-HRMS spectrum of each of these compounds displayed, in addition to the [M + H] + ion (apo form, not Fe-bound), an ion at 52.9117 amu difference [M + 56 Fe − 3H] + , corresponding to their Fe-adduct, thus, suggesting the presence of Fe-chelating compounds. Six nodes were identified as synechobactins (Table 1), i.e., the hydroxamate-type siderophores. Synechobactins have a backbone consisting of citric acid, linked to two 1,3-diaminopropane units. The first diaminopropane unit is N-acetylated and N-hydroxylated, forming the hydroxamate group. The second diaminopropane unit is N-hydroxylated and N-acylated, with a fatty acid tail that varies in length and degree of unsaturation [23]. The molecular mass of 6, corresponding to the molecular formula of C 30 H 55 O 9 N 4 + , was 2 amu (2 hydrogen atoms) less than that of synechobactin C 16 (5), suggesting the presence of a double bond. The additional unsaturation was allocated to the fatty acid tail, the peak at m/z 361.1706 in the MS 2 spectrum of 6 resulting from the neutral loss of the unsaturated fatty acid C 16 H 30 O 2 (254.2235 amu). The position and the configuration of the double bond remained unassigned. To the best of our knowledge, the only reported synechobactin structure in which an unsaturated acyl chain is present is rhizobactin (C 10 ) [25]; therefore, we propose that synechobactin C 16 Compounds (1)(2)(3)(4)(5)(6)(7)(8) share the same dihydroxamate-citrate moiety, being different only in the structure of the fatty acid residue; compounds 9-12 are modified in the polar part. Both the cleavages from the pseudomolecular ions' peaks at m/z 517.3212 (9) and 545.3525 (10) of dodecanoic and tetradecanoic acid, respectively, are observed for synechobactin A and C 14 , generating the fragment ion C 12 H 21 O 6 N 4 + (m/z 317.1459), in consistence with the non-acetylated hydroxamate/citrate backbone. This was confirmed by the presence of the peak at m/z 429.2601, in the MS 2 spectrum of 9, due to the loss of the 3-(hydroxyamino)propan-1-amine (88.0634 amu, C 3 H 8 ON 2 ) unit, which was also present in the MS 2 spectrum of synechobactin A, due to the loss of the N-(3-aminopropyl)-Nhydroxyacetamide residue (132.0894 amu). The 15.9948 amu difference between synechobactin C 14 (4) and the ion at m/z 573.3839 (compound 11) accounts for an oxygen atom. The loss of tetradecanoic acid from the pseudomolecular ion of 11 leads to the peak at m/z 345.1772 (C 14 H 25 O 6 N 4 ), indicating that the oxygen atom is missing in the dihydroxamatecitrate portion of the molecule. The peak at m/z 328.1508 (C 14 H 22 O 6 N 3 ), in both the MS 2 spectra of synechobactin C 14 (4) and deoxyC 14 (11), corresponds to the fragment (citryl)-N-(3-aminopropyl)-N-hydroxyacetamide, pointing to the lack of a hydroxyl group on the nitrogen of the other hydroxamate. Likewise, in compound 12, the dehydrated form of deoxy synechobactin C 14 , dehydration occurs on the second hydroxamate unit. The thirteenth node of the synechobactins' cluster (m/z 658.2852, C 28 H 50 O 10 N 4 Fe + ) is the iron-bound ion of synechobactin oxyC 14 (7).

Anabaena flos-aquae UTEX 1444: Strain, Cultivation and Extraction
The genus Anabaena BORY ex BORN. et FLAH. 1888 encompasses filamentous, nonbranched species, which differ in the form of cells, coiling of trichomes, shape and size of akinetes [26]. Anabaena species exhibit a wide range of strain-specific variability, and environmental conditions mainly affect cell dimensions and size of heterocysts [27].
The species A. flos-aquae Brébisson ex Bornet et Flahault 1888, is a free-floating organism, whose coiled filaments can be solitary or irregularly assembled, with narrow mucilaginous sheath. Cells are usually spherical to barrel-shaped, whereas akinetes can be cylindrical or elliptical and heterocysts usually spherical [28]. A. flos-aquae is diffused in freshwater environments worldwide and is responsible of blooms that can have noxious effects on other organisms, due to the excretion of toxic metabolites, as anatoxins [29].
From a taxonomical point of view, A. flos-aquae is a combined morphological group, in that an alternation of regularly and irregularly coiled filaments is observed for the same strain in laboratory cultures; moreover, vegetative cell shape and size, along with heterocysts and akinetes dimensions, were largely variable among different populations [30].

LC-HRMS Analyses and Molecular Networking
LC-HRMS experiments were performed using a Thermo LTQ Orbitrap XL highresolution ESI mass spectrometer (Thermo Fisher Scientific Spa, Rodano, Italy) coupled to an Agilent model 1100 LC system (Agilent Technologies, Cernusco sul Naviglio, Italy), which included a solvent reservoir, in-line degasser, binary pump, and refrigerated autosampler. A 5-µm Kinetex C18 column (50 × 2.10 mm), maintained at room temperature, was eluted at 200 mL min −1 with H 2 O (supplemented with 0.1% HCOOH) and MeOH, using gradient elution. The gradient program was as follows: 10% MeOH for 3 min, 10%→100% MeOH for 30 min, 100% MeOH for 7 min. Mass spectra were acquired in positive ion detection mode. MS parameters were a spray voltage of 4.8 kV, a capillary temperature of 285 • C, a sheath gas rate of 32 units N 2 (ca. 150 mL/min), and an auxiliary gas rate of 15 units N 2 (ca. 50 mL/min). Data were collected in the data-dependent acquisition (DDA) mode, in which the first, the second, up until the tenth most intense ions of a full-scan mass spectrum were subjected to high-resolution tandem mass spectrometry (HRMS/MS) analysis. The m/z range for data-dependent acquisition was set between 100 and 2000 amu. HRMS/MS scans were obtained for selected ions with CID fragmentation, isolation width of 2.0, normalized collision energy of 35, Activation Q of 0.250, and activation time of 30 ms. Data were analyzed using Thermo Xcalibur software.
Raw files were imported into MZmine 2.53 open-source software [14]. The mass detection was performed on raw data and exact masses with mass level 1 and centroided masses with mass level 2, by keeping the noise level at 1000. Chromatograms were built using an ADAP module with a minimum height of 1000, and m/z tolerance of 0.001 (or 5 ppm). Peak alignment was performed using the Join aligner algorithm (m/z tolerance at 0.005 (or 5 ppm), absolute RT tolerance at 0. were filtered out by setting the maximum relative height at 100%. Peaks without associated MS/MS spectra were finally filtered out from the peak list. Clustered data were then exported to .mgf file for GNPS, while chromatographic data including retention times, peak areas, and peak heights were exported to a .csv file.
A Feature-Based Molecular Network was generated on GNPS' online platform [15], with the following parameters: the parent mass tolerance and MS/MS fragment ion tolerance were set both at 0.05 Da, the cosine score at above 0.5, and matched peaks above 5. Spectra were retained only if the nodes appeared in each other's respective top 10 most similar nodes. The spectra in the network were then searched against GNPS spectral libraries using a cosine score above 0.7 and at least 6 matched peaks. The molecular network was visualized using Cytoscape software [16].

Conclusions
It is well known that cyanobacteria have a unique adaptive character due to their metabolism capability to react to adverse environmental conditions. They represent a large group of bioagents, less explored as bioresources that produce high-value natural products with biotechnological and ecological relevance. This paper reports on our study on the cyanobacteria Anabaena flos-aquae strain UTEX 1444, as a promising sustainable bioresource of bioactive molecules, i.e., siderophores. We explored metabolome variations when UTEX 1444 was cultivated in the condition of iron deficiency and iron overload. As presumed, the cultivation of the strain in conditions of iron deficiency revealed its capability to produce siderophores. In our procedure, to obtain a fast dereplication of #1-6 organic extracts, molecular networking analyses of MS/MS data were successfully used. Comprehensive FBMN, featuring #1-6 metabolites' relative abundance comparison, immediately disclosed the presence of a cluster composed of metabolites, belonging only to the zero iron (III) culture and, therefore, allowed us to easily and quickly pinpoint the siderophore cluster; moreover, molecular networking allowed for a fast detection of synechobactins and new variants, thanks to their typical fragmentation pattern. HRMS/MS analysis and the fragmentation pattern of synechobactin A, C 16 and the new synechobactin C 16:1 are reported as a useful diagnostic way to detect them in any extract. The structure and the kind of cyanobacterial siderophores were determined in only a few previously published papers [7].
Together with the five known synechobactins, six new variants have been identified, showing unusual modifications occurring in the nature of the acyl chain (unsaturated, hydroxylated) and in the hydroxamate-citrate backbone. To our best knowledge, this is the first report unveiling the biosynthetic capability of UTEX 1444 to produce a large array of synechobactins. It is interesting to note that UTEX 1444 produced 11 variants, beside schizokinen, showing the ability to incorporate a variety of saturated and unsaturated fatty acids, likely as a final biosynthetic step. Modifications also occur in the hydroxamate-citrate backbone, extending the chemical variants of the synechobactins known so far.
Considering the potential high impact of siderophores production as ferric ion scavengers, as well as toxic metal chelators, and then, the large array of applications in many different fields, from ecological research to drug discovery [5][6][7], our future studies will be focused on making UTEX 1444 a suitable model organism for a scale-up production of siderophores. This report, encompassing methodological improvements in siderophore production, characterization and detection through the dereplication of extracts, using a powerful bioinformatic approach, is a sustainable contribution to quest natural molecules that improve environmental and human health. Following EU direction, this study follows the "Do no significant harm" requirement and overcomes the "supply" problem that is the main bottleneck in the study of novel bioactive molecules from marine organisms.


Magnetic Drug Delivery System: New Hope for Cancer Patients

1Department of Pharmacology, MET’s Institute Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra, India, 422003. 2Department of Quality Assurance Techniques, MET’s Institute Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra, India, 422003. 3Department of Pharmaceutical Chemistry, MET’s Institute Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra, India, 422003. 4Department of Pharmaceutics, MET’s Institute Pharmacy, Bhujbal Knowledge City, Adgaon, Nashik, Maharashtra, India, 422003.

In conventional drug delivery system for cancer treatment anti-cancer drug given intravenously this drug may accumulate in cancerous cells which may contain a large number of leaking blood vessels, so due to accumulation of this drug it may also adversely affect on healthy tissue and show lots off side effect which is the biggest disadvantage of conventional drug delivery system. Some of the nanomedicines show high accumulation of drug on tumour site due to enhanced permeability and retention (EPR) effect it offers increases vascular permeability which is beneficial but EPR effect may vary from person to person and disease condition so it is insufficient to match challengeable and complicated tumour microenvironment. In a magnetic drug delivery system, we can manipulate a drug containing magnetic moment and block it to act on the only cancerous cell so the drug will not affect healthy tissue and these results in improvement of efficacy and reduce dosing of the drug (Fig 1). Magnetic drug delivery system can be used in the treatment of cancers, nervous system disorders, sudden sensorineural hearing loss, gene therapy, etc.
The magnetically modulated drug is also a good agent for Magnetic resonance imaging (MRI) and it gives the good advantage of diagnosis and treatment of disease by the single agent (Table 1) also used in molecular biology, cell isolation and purification, Hyperthermia, and radioimmunoassay 1 . Apart from advantages MDDS has several disadvantages such as highcost technique, required specialized magnets for targeting, trained personnel required to perform drug therapy, cannot be used for cancerous cells that situated in organs deep inside the body but recent advancements are done in which magnet is implanted near the targeted area to overcome this problem 2 . Aim of work is to gain focus on magnetic drug delivery system, during the last few decades, magnetic drug delivery systems have been a popular approach for site-specific targeting of various pharmacological agents. It avoids the reticuloendothelial system and accurately sends the medications to the target with the help of a magnetic field. History Magnetic drug delivery system is comparatively newer technique Gilchrist in 1956 publish seminar that explains after injections of magnetism inducing particles in lymph nodes near surgically removed tumour inductive heating of lymph nodes takes place. In 1963 Meyers explain how they successfully guided and block small iron particles in dog leg veins with help of a horseshoe magnet 1

Magnetic nanoparticle
Particles that have a size of less than 1 micrometer and can be manipulated using a magnetic field are magnetic nanoparticles. Magnetic nanoparticles have lots of advantages such as high stability, more carrier capacity can incorporate both hydrophilic and hydrophobic drugs and controlled release can also be possible which results in an increase in bioavailability and reduction in dosing frequency 4 , Magnetic nanoparticles also have a low sedimentation rate and higher tissue diffusion 5 . Magnetic nanoparticles did not possess any immunogenicity or virulence property 6 .
Magnetic material generally has a multidomain structure but nano-sized magnetic particles have one domain structure and their magnetism property changes to paramagnetic nature 7 . Hence due to nano size, magnetic nanoparticles show super magnetism which allows easy targeting to cancerous cells.
Corona formation 8 i.e. when nanoparticles come in contact with biological fluid they absorb proteins and lipids on their surfaces is a limitation of magnetic nanoparticles 9 . Magnetic nanoparticles generally composed of 1) Magnetic core 2) Coating 3) Functional group on the surfaceby use of suitable surfactant surfaces of nanoparticle make functionalized by making them hydrophilic 10 . Coating is done to prevent aggregation and prevent interaction with system environment. Nanoparticles which contain metal oxides Fe 2 O 3 and Fe 3 O 4 mostly used( Table 2). Magnetic nanoparticles can be prepared by ionic and non ionic method 11

Preparation of magnetic nanoparticles Green synthesis of magnetic nanoparticles
Green nanotechnology is very efficient as it helps to reduce or eliminate toxic substances to restore the environment. Synthesis of nanoparticles from the plant is currently under development process. Green synthesis of a magnetic nanoparticle is a safe, non-toxic, and environment-friendly process. Inactivated plant tissue, plant exudates, plant extract, and other parts of living plant is used in green synthesis for the production of magnetic nanoparticles. The biological method can also be used for the preparation of magnetic nanoparticles in which micro-organisms, fungi, and enzymes are used but a preparation of nanoparticles by plant or plant extract is preferred over other biological processes as it eliminates the elaborate work involved in microbial culture maintenance.
Aw w a d A . M . a n d S a l e m N . M . recommended a method for the synthesis of magnetic nanoparticles in a single-step reaction through green synthesis. Extract of carob leaf, Ferric chloride tetrahydrate, ferric chloride hexahydrate, Sodium hydroxide used in the experiment. By this process, magnetic nanoparticles are obtained at a low temperature of about 80-85°C having 4 to 8 nm size and good monodispersible properties.

Precipitation from solution
Precipitation of product from solution for nanoparticle preparation is one of the oldest methods. In this method metal precursors are dissolved in a solvent such as water and an insoluble solid is generated by the addition of precipitating agent. By homogeneous precipitation reaction, uniform particles are synthesized.

Co-precipitation
This is the most widely used method for the synthesis of magnetic nanoparticles. Aqueous salt solutions are used for the preparation of magnetic nanoparticles by base addition under an inert atmosphere at room temperature or high temperature. Spherical magnetic nanoparticles in solution is synthesized by two approaches: Partially oxidizing Fe(OH)‚ suspension with help of the different oxidizing agent and aging stoichiometric mixture of ferric and ferrous hydroxides in aqueous media which forms spherical homogeneous magnetic nanoparticles. Salt used, pH value, ferric and ferrous ion ratio, reaction temperature, ionic strength of media decides the size and shape of nanoparticles.

Microemulsion
It is isotropic, thermodynamicallymeas stable, transparent dispersion of two immiscible liquids such as oil and water stabilization is done by use of surfactant. At the water and oil interface surfactants form a monolayer, with hydrophobic tails in the oil phase and hydrophilic head dissolving in the aqueous phase. Advantages of using the microemulsion technique are the use of simple equipment, which can be used to synthesize a large variety of materials and can control particle size and composition. Nanoparticles produced by this technique are small in size with higher saturation magnetization. The structure and type of surfactant decides the properties of nanoparticles.

Polyol method
The polyol method is used for the synthesis of uniform size nanoparticles which can be used in magnetic resonance imaging (MRI). In this method,  Diagnosis and treatment by single agent not possible Diagnosis and treatment can be possible by single agent 5.
Cost is Low Cost is high fine metallic particles are synthesized by reducing dissolved metallic salt and precipitating metals from the solution including polyol. Thermal decomposition of organic precursors Samples with good size control, good crystallinity, and narrow size distribution have yielded after decomposition of iron (Fe) precursors in presence of hot organic surfactant. Thermal decomposition synthesized nanoparticles with the high level of monodispersity.

Hydrothermal method
The solvothermal method is another name for the hydrothermal method. By this method, magnetic nanoparticles and ultrafine powders can be synthesized. This is one of the successful method to grow crystals of different material.

Chemical vapour deposition
Chemical vapour deposition is the method that can produce a wide range of materials and can take the benefits of a large database of chemistries that have been developed for this process. In this method, the particle size distribution of nanoparticles can be controlled by controlling the mixing of cold gas with hot gas carrying evaporated material.

Spray pyrolysis
In spray pyrolysis solid is prepared by spraying solution on reactors where the solvent evaporates from aerosol droplets with condensation of solute within droplet after that drying and thermolysis of the particle at high temperature is done. Maghemite nanoparticles start with Fe3+ salt and organic compound acts as reducing agent generated by most of the pyrolysis process.

Laser pyrolysis
It is a pyrolysis process in which laser energy is used for the synthesis of nanoparticles. This method allows rapid cooling and highly localized heating as compared to heating gases in a furnace. In this method flowing mixture of gases is heated with a carbon dioxide laser which initiates a chemical reaction.

Sonochemical reaction
Novel materials having unusual properties are synthesized by the sonochemical reaction. In sonochemical reaction, ultrasound energy is used. Nanopowder prepared by this method is generally amorphous, porous, and agglomerated 12 (Fig 2). Applications of magnetic nanoparticles a. Magnetic nanoparticles used as the carrier in conjugation with tetrahedral antibodies and chemotherapeutic agent 13 . b. Antibody linked fluorescent MNP used for target imaging and treatment of GIT cancer 14 . c. MNP can be used in hyperthermia and magnetofection. d. MNP can replace fluorescent and optical labels in biosensors 15 . e. For DNA absorption MNP coated with meso-2, 3-dimercaprosuccinic acid containing carboxylic acid group used 16 . f . For Escherichia coli detection nanoparticles with iron oxide gold core-shell used 17 . g. Human chorionic gonadotropin can be detected by SPR sensor chip which is the combination of MNP with antibodies. h. Signal of fluorescence can be increased by nanoparticles 18 . i. MNP can be used as the vector for gene transportation 19 . j. MNP can be used in tumour thermotherapy as magnetic nanoparticles can produce the thermal effect in changing magnetic field 6 .

Microsphere
Magnetic microspheres are proteins or polymers having particle size of 1-100 micrometer and it is biodegradable 20 . Magnetic microspheres are small enough as can circulate through capillaries without forming embolic occlusion but can able to entrapped in microvessels and dragged in adjacent tissues by using magnetic field 21,22 .Without magnetic carrier major portion of the drug reaches to RES (Reticuloendothelial system) organs while drug with magnetic carrier all drug reaches to targeted tissue (Fig 3) 23 . Magnetic microsphere can be used for the controlled release of drugs, antibodies, vaccines, hormones, etc. Lupron Depot®, Nutropin Depot® are products of polymeric microsphere 22 .By altering the size of the microsphere, changing drug content, altering magnetite content, changing hydration state, and changing drug release characteristics of carrier rate and amount of drug release can be controlled 23 .
Loaded magnetic microsphere injected in the blood vessel by using 18 or 16 number needle guided by the magnet and in a very short period, they gathered at the targeted site where they emit radiations to kill cancerous cells. There are two types of microsphere one is the therapeutic microsphere which is used for the treatment of disease and the other one is the diagnostic microsphere which is used for diagnosis e.g. Microsphere used for liver metastasis imaging, bowel loops, and other abdominal structures can distinguish by using magnetic microsphere 24

Various methods of preparation are a. Solvent evaporation
Solvent evaporation technique used to prepare polymer encapsulated microsphere. The auxiliary solution was prepared by the addition of drug, magnetite, and polymer in a volatile organic solvent. The resulting auxiliary solution is homogenized and stirred at a 22-30°c temperature which forms a magnetic microsphere that is separated by centrifugation and stored at 4°C 25,26 .

b.Multiple emulsion method
In this method, w/o/w emulsion is formulated. Aqueous protein solution containing the active ingredient is dispersed in the lipophilic phase. The polymer solution is generally a continuous phase that encapsulates protein present in the dispersed aqueous phase, before adding to an aqueous solution of polyvinyl alcohol primary emulsion subjected to homogenization or sonication which forms multiple emulsion, and then solvent evaporation is done to form magnetic microsphere 27 .

c. Phase separation emulsion polymerization
The aqueous solution of polymer, drug, and magnetite is added in vegetable oil then emulsification is done by stirring heating is done at 100-150°c for stabilization of emulsion, then with the continuous stirring cross-linking agent is added which forms magnetic microsphere which is separated by washing 25 .

d. Dispersion co-polymerization
This method involves the reaction of various monomers at the interface between two immiscible liquid phases to form a film of polymer that envelopes the dispersed phase. In this method, two monomers are used one monomer dissolved in a continuous phase while the other monomer dispersed in a continuous phase. E.g. by dispersion co-polymerization of styrene and polyethylene oxide vinyl benzyl (PEO-VB) amphiphilic magnetic microsphere formed having a size range 5-100 micrometer 25 .

e. Hot melt microencapsulation
In hot melt microencapsulation initially, heating of polymer is done and then added into solid particles of the drug, this mixture suspended in an immiscible solvent, continuous stirring is done after that heating is carried out heating temperature should be 5°c more than the melting point of the polymer. Polymer particles solidified by cooling and microsphere is formed which then washed with petroleum ether 26 .

f. Microwave-assisted preparation of magnetic albumin microsphere
This method is faster than the traditional method and produces comparatively small particles. This method is mostly used for the preparation of magnetic protein microsphere 28 . Factors that influence properties of microsphere

Choice of solvent
The solvent should be chosen such that it can dissolve the selected polymer, more volatile and less toxic

Antifoaming agent
Foaming is one of the major problems which can disturb the formation of microspheres antifoaming agents such as dimethicone, spans are used to avoid foaming.

Surfactant
Surfactants generally stabilize emulsion by reducing surface tension and avoiding coalescence and agglomeration. Methylcellulose, tween, span, sodium dodecyl sulphate, etc. is used as a surfactant. Application of magnetic microsphere a. Magnetic microspheres used in immobilization of enzymes, isolation of cells, purification of proteins 25,29 . b. Mitoxantrone, paclitaxel, doxorubicin likes drugs can be incorporated in microsphere and used in cancer treatment 30 . c. For a preclinical study to treat liver and brain tumour magnetic microsphere labelled with Rhenium-188 and Yttrium-90 used 31 . d. Stem cell extraction and bone marrow purging possible by magnetic microsphere 32 . e. For treatment of cancer by localized hyperthermia magnetic microsphere with paclitaxel and cisplastin used 33 . f. Magnetic beads which are coated with streptavidin are used for the detection of bacteria 34 .

Magnetic Liposome
Liposomes are spherical vesicle that contains a minimum one lipid bilayer which contains cholesterol and natural non-toxic phospholipid. Liposome is advantageous as it has greater bioavailability, particle size can be adjustable, Hydrophilic, as well as hydrophobic drugs, can be incorporated, Surface modification can be possible which can help to pass biological barrier 35 . In the case of magnetic liposome magnetite is an additional component prepared by aentrapment of ferrofluid within the core of the liposome, which helps to guide the liposome to the targeted site with help of the magnet. Thermosensitive liposome release drug after heating by EMR 1 . There are two types of magnetic liposomes one which encapsulates metal-oxide ions in the aqueous layer while the other contains metal oxide enveloped in lipid layer 36 . Application of magnetic liposome: 1. Magnetic liposome use as cell or tissue sitespecific accumulation drug delivery. 2. Magnetic liposome has specific functions in magnetic-related characteristics such as contrast agent, magnetic-targeted ability, and heating generation

Magnetic microbubble
Magnetic microbubble is responsive to applied magnetic field changes and can be visible by ultrasonography used for magnetic drug delivery. Recently for the formulation of magnetic microbubble suspension of protamine functionalized microbubble mix with a suspension of heparinized NP 41 . Magnetic microbubble guided to tumours and imaging is done by ultrasonography. Focused ultrasound is used to collapse microbubble and then the drug is released 37 .

Magnetic Microcapsule
Magnetic microcapsule which is used in vivo and in vitro consisting of poly (allylamine hydrochloride) and poly (sodium 4-styrenesulfonate) and developed by LbL deposition10.This is the most promising drug delivery system with remote navigation by the magnetic field. Mesoporous mushroom (Agaricusbisporous) is used to prepare microcapsules called iMushbots. IMushbots show higher drug retaining properties at alkaline pH i.e. in blood while easy drug release in acidic medium (cancerous cells) 38 .

ConCLuSion
Magnetic delivery is very useful in treating life-threatening diseases; Drug targeting is very easy with the magnetic drug delivery system which is the biggest advantage. Magnetic drug delivery is the latest technology that received attention in the 1990s.In the early 20th century Paul Ehrlich proposed the Magic bullet concept i.e. drugs reaches the right site in the body at right time at the right concentration and magnetic drug delivery systems fulfil all these objectives. It is a challenging area for researches carried out in the future so more researches, long-term toxicity study, and characterization should be done for continuous improvement in the field.


s2fieldsofstudy: Biology Engineering

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HDAC6 modulates myofibril stiffness and diastolic function of the heart

Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases.

Introduction
Stiffness of the heart is categorized as either active or passive. Active stiffness is dependent on actomyosin cross-bridge interactions, while passive stiffness is determined by myocardial extracellular matrix and titin (1). Modulation of titin compliance is linked to human cardiac disease (2), with decreased titin-based passive stiffness observed in patients with systolic dysfunction and dilated cardiomyopathy (DCM) (3)(4)(5)(6), and titin stiffening associated with diastolic dysfunction (DD) and heart failure with preserved ejection fraction (HFpEF) (7)(8)(9)(10). Increased compliance of titin in DCM impairs active contraction and sarcomere length-dependent tension generation (the Frank-Starling mechanism) (11,12), while excessive titin stiffening in HFpEF diminishes ventricular filling during diastole (2,8). Given the central role of titin in the control of cardiac homeostasis and disease, there is intense interest in developing therapeutic modalities with the capacity to negatively or positively tune titin compliance.
Titin, a modular protein that spans from the Z-disk to the M-band of the sarcomere, is integral to the regulation of myofibril passive stiffness. Titin is initially expressed as a fetal isoform of N2BA. In adults, RBM20-mediated splicing favors the expression of a shorter N2BA, which is gradually replaced by the even shorter N2B (Supplemental Figure 1; supplemental material available online with this article; https://doi.org/10.1172/JCI148333DS1) (13)(14)(15)(16). Both isoforms consist of an amino-terminal Z-disk-binding region followed by an I-band spring region, the latter of which is the primarily subjected to differential splicing. Both titin isoforms harbor a common N2B element within the spring region. However, the spring region of the N2BA isoform contains more immunoglobulin-like (Ig-like) domain segments and a longer PEVK element, so named because it is rich in proline, glutamic acid, valine, and lysine residues. As such, with an extended spring region, the N2BA isoform is more compliant than the adult N2B isoform of titin. Other domains that are common to both isoforms of titin comprise the myosin-binding A-band, and the M-band, which binds myomesin (Supplemental Figure 1) (2). Altered titin N2BA/N2B expression ratio is associated with certain forms of heart failure (HF) in humans (2).
Passive stiffness of the heart is determined largely by extracellular matrix and titin, which functions as a molecular spring within sarcomeres. Titin stiffening is associated with the development of diastolic dysfunction (DD), while augmented titin compliance appears to impair systolic performance in dilated cardiomyopathy. We found that myofibril stiffness was elevated in mice lacking histone deacetylase 6 (HDAC6). Cultured adult murine ventricular myocytes treated with a selective HDAC6 inhibitor also exhibited increased myofibril stiffness. Conversely, HDAC6 overexpression in cardiomyocytes led to decreased myofibril stiffness, as did ex vivo treatment of mouse, rat, and human myofibrils with recombinant HDAC6. Modulation of myofibril stiffness by HDAC6 was dependent on 282 amino acids encompassing a portion of the PEVK element of titin. HDAC6 colocalized with Z-disks, and proteomics analysis suggested that HDAC6 functions as a sarcomeric protein deacetylase. Finally, increased myofibril stiffness in HDAC6-deficient mice was associated with exacerbated DD in response to hypertension or aging. These findings define a role for a deacetylase in the control of myofibril function and myocardial passive stiffness, suggest that reversible acetylation alters titin compliance, and reveal the potential of targeting HDAC6 to manipulate the elastic properties of the heart to treat cardiac diseases. HDAC6 modulates myofibril stiffness and diastolic function of the heart Here, we demonstrate that the zinc-dependent deacetylase histone deacetylase 6 (HDAC6) tightly regulates cardiac myofibril compliance, and we provide evidence to suggest that this effect is mediated by alterations in titin acetylation. HDAC6 inhibition leads to myofibril stiffening, while enhancement of HDAC6 activ ity increases myofibril compliance, both through mechanisms that are dependent on the PEVK element of titin. HDAC6 is currently being aggressively pursued as a therapeutic target for multiple indications, including cancer and neurodegenerative diseases, by virtue of its ability to regulate processes such as protein turnover and mitochondrial transport (20). Our data suggest that future studies of HDAC6 inhibitors in the clinic should incorporate evaluation of cardiac function, particularly diastolic performance. Beyond this, the findings presented here highlight a path toward the development of innovative treatments for HF, with HDAC6 inhibition and subsequent myofibril stiffening potentially ameliorating cardiac diseases marked by excessive titin compliance and systolic dysfunction, and HDAC6 gain of function benefiting patients with DD that is associated with titin stiffening.
Another mechanism for regulation of titin compliance is through differential phosphorylation, with the spring elements of titin (N2B and PEVK) serving as hotspots for phosphorylation by kinases such as protein kinase A (PKA), protein kinase G (PKG), protein kinase C (PKC), extracellular signal-regulated kinase (ERK), and Ca 2+ /calmodulin-dependent protein kinase II (CaM-KII) (Supplemental Figure 1) (8). From a therapeutic perspective, most effort has focused on restoring titin phosphorylation by stimulating PKG activity via phosphodiesterase 5 (PDE5) inhibition or soluble guanylyl cyclase (sGC) activation as an approach to decrease passive stiffness of the heart to improve relaxation in patients with HFpEF (17,18).
Other posttranslational modifications of titin, including oxidation, glutathionylation, and sulfenylation, have been linked to the regulation of myofibril stiffness. Additionally, deacetylation of titin by NAD-dependent sirtuins was recently found to be associated with increased titin compliance (19). However, the degree to which reversible acetylation on lysine residues regulates titin-mediated passive stiffness remains unclear. (F) Myofibril resting tension-to-sarcomere length curves. (G) Myofibrils were treated with the myosin ATPase inhibitor butanedione monoxime (BDM; 50 mM) before assessment of resting tension at the given sarcomere lengths. For F and G, data are presented as mean ± SEM, fitted by third-order polynomials, from 4 animals per group, with 6-8 myofibrils per mouse analyzed. and residual cross-bridge binding, was elevated in HDAC6-KO mice compared with WT controls ( Figure 1E).
To expand the evaluation of the impact of HDAC6 on cardiac stiffness, myofibrils from WT and HDAC6-KO hearts were subjected to stepwise extension ex vivo, and concomitant alterations in resting tension were quantified to yield resting tension-to-sarcomere length curves (23). HDAC6 deletion resulted in a resting tension-to-sarcomere length curve in comparison with WT controls ( Figure 1F). A similar effect was observed using myofibrils treated with the myosin ATPase inhibitor butanedione monoxime (BDM) ( Figure 1G), ruling out the possible contribution of residual binding of cross-bridges to this phenotype, and further suggesting that HDAC6 deletion diminishes titin compliance.
To determine whether HDAC6 catalytic activity regulates myofibril stiffness, cultured adult rat ventricular myocytes (ARVMs) were treated for 24 hours with the HDAC6-selective inhibitor tubastatin A (20); ITF2357, which inhibits HDAC6 as well as several other zinc-dependent HDACs; or vehicle control ( Figure 2A). Consistent with the findings from KO hearts, myofibrils from ARVMs exposed to tubastatin A exhibited elevated resting tension ( Figure 2B). Surprisingly, ITF2357 had no effect

Results
HDAC6 loss of function leads to titin stiffening. Reversible acetylation of sarcomeric proteins is emerging as a mechanism for posttranslational regulation of cardiac function (21), but the roles of specific HDAC isoforms in the control of myofibrillar proteins remain unclear. Previously we demonstrated that mice lacking HDAC6, which serves prominent roles in the cytoplasm by deacetylating cytoskeletal proteins, have apparently normal cardiac function at baseline, but are protected from systolic dysfunction elicited by chronic angiotensin II administration or transverse aortic constriction (22). To further address the possibility that HDAC6 functions in the cytoplasm of cardiomyocytes to regulate mechanical properties of sarcomeres, bundles of sarcomeres (myofibrils), obtained from homogenized left ventricles (LVs) of WT and HDAC6-KO mice, were mounted on a microtool attached to a piezo motor and a cantilevered force probe, and their mechanical properties were quantified ex vivo ( Figure 1A). Myofibrils from HDAC6-deficient mice generated force in response to Ca 2+ and relaxed upon Ca 2+ removal equivalently to those from hearts of WT mice (Figure 1, B-D, and Supplemental Figure 2). In contrast, myofibril resting tension, which is a measure of titin compliance negative control (Ad-β-gal). Immunoblotting confirmed efficient expression of ectopic HDAC6 in the cultured adult cardiomyocytes (Supplemental Figure 3A). In agreement with prior findings (26,27), deacetylase domain 2 of HDAC6 was required for deacetylation of endogenous α-tubulin (Supplemental Figure 3, A and B), establishing the validity of this cell-based, ectopic expression system. Consistent with a role for HDAC6 in controlling myofibril function, confocal imaging revealed a pool of the enzyme that colocalized with sarcomeric α-actinin at the Z-disk in cardiomyocytes, independently of its catalytic activity ( Figure 3B and Supplemental Figure 3C). To determine whether HDAC6 gain of function alters passive stiffness of cardiomyocytes, ARVMs were infected with the adenoviruses for 72 hours, and myofibrils were subsequently isolated for evaluation of mechanics ( Figure 3C). Strikingly, ectopic expression of HDAC6 dramatically increased the compliance of cardiac myofibrils in a manner dependent on the catalytic activity of deacetylase domain 2 ( Figure  3D and Supplemental Figure 3D). Ex vivo incubation of purified rat myofibrils with recombinant HDAC6, but not HDAC2, reduced titin stiffness ( Figure 4, A and B), establishing the ability of HDAC6 to directly and selectively modulate sarcomere function; HDAC2 was chosen as a control for specificity because of our prior demonstration that the enzyme associates with cardiac myofibrils to regulate relaxation (21). Recombinant HDAC6 also increased compliance of human myofibrils obtained from nonfailing donor LV explants, illustrating a conserved ability of this deacetylase to control cardiomyocyte passive stiffness in higher mammals ( Figure 4C).
The PEVK region of titin is required for HDAC6-mediated modulation of myofibril stiffness. To begin to determine whether HDAC6-mediated alterations in myofibril stiffness are governed via titin, recombinant HDAC6 was incubated with myofibrils obtained from adult mouse hearts from WT mice or mice in which of myofibril stiffness. HDAC6 is a tubulin deacetylase (24), and pharmacodynamic assessment of tubulin acetylation as a marker of HDAC6 inhibition confirmed that tubastatin A and ITF2357 inhibited HDAC6 equivalently in ARVMs ( Figure 2, C and D). Thus, enhanced myofibril stiffness is only observed upon selective inhibition of HDAC6. These findings agree with the prior demonstration that ITF2357 ameliorates rather than exacerbates DD in murine models (21,25).
HDAC6 gain of function leads to decreased titin stiffness. Unlike other HDACs, HDAC6 contains tandem deacetylase domains ( Figure 3A). To further address the function of HDAC6 in the heart, ARVMs were infected with adenoviruses expressing wildtype HDAC6 (Ad-HDAC6 WT), derivatives of the enzyme harboring amino acid substitutions that abolish catalytic activity (Ad-HDAC6 H216A, H611A, or H216/611A), or a β-galactosidase  a portion (amino acids 12717-12998) of the PEVK element and an adjacent Ig-like domain of titin were deleted by homologous recombination ( Figure 4D) (28); these PEVK residues are expressed in all titin isoforms. As with samples obtained from rat and human hearts, recombinant HDAC6 efficiently reduced the stiffness of mouse cardiac myofibrils ( Figure 4E). Remarkably, myofibrils from mice lacking 282 amino acids of the PEVK/Ig-like region of titin were com-pletely resistant to HDAC6 ( Figure 4F). Notably, the acetylation state of cardiac α-tubulin was unaffected by deletion of the PEVK domain, arguing against an indirect role for this region of titin in the control of HDAC6 activity (Supplemental Figure 4, A and B).
Next, we sought to determine whether myofibril stiffening upon HDAC6 inhibition is dependent on the PEVK domain of titin. Adult mouse ventricular myocytes (AMVMs) were obtained Mean + SEM is shown; *P < 0.05 vs. untreated myofibrils based on 1-way ANOVA with Tukey's multiple-comparison test. (D) Myofibril resting tension-to-sarcomere length curves. Data are presented as mean ± SEM, fitted by third-order polynomials. Myofibrils from 3 nonfailing human hearts were each treated with vehicle, recombinant PKCα, or recombinant PKCα followed by recombinant HDAC6; 6-8 myofibrils per treatment were analyzed and averaged per heart. (E) Immunoblotting was performed with an anti-PKC substrates antibody and solubilized proteins from myofibrils treated as indicated. (F) Titin was immunoblotted with antibodies specific for phospho-S11916 (S11878 in human) and phospho-S12037 (S12022 in human) in the PEVK domain, as well as an antibody against the titin Z1Z2 element to assess total titin levels.
from WT and PEVK-KO animals and cultured in the absence or presence of tubastatin A before isolation of myofibrils for mechanical measurements ( Figure 4G). In line with data obtained with ARVMs, pharmacological inhibition of HDAC6 with tubastatin A led to an increase in myofibril resting tension in WT AMVMs (Figure 4H). However, the HDAC6 inhibitor failed to augment passive stiffness of PEVK-KO myofibrils; PEVK-KO myofibrils had higher basal resting tension than WT controls ( Figure 4I), which is consistent with the previously defined role for this region of titin in the regulation of myofibril compliance (28). Analysis of resting tension-to-sarcomere length curves confirmed that tubastatin A-mediated stiffening of myofibrils was dependent on the PEVK region of titin ( Figure 4I). Together, these findings define a critical role for the PEVK spring element of titin in HDAC6-mediated regulation of cardiomyocyte passive stiffness.
HDAC6 reverses PKC-mediated titin stiffening in human myofibrils. PKC-dependent phosphorylation of the PEVK element of titin leads to myofibril stiffening (29), while PKA-and PKG-mediated phosphorylation of the adjacent N2B region increases titin compliance ( Figure 5A) (30,31). Immunoblotting of mouse LV homogenates using phospho-specific antibodies failed to reveal an impact of HDAC6 deletion on PEVK or N2B phosphorylation (Supplemental Figure 5, A and B), suggesting that HDAC6 regulates titin stiffness by directly deacetylating the protein as opposed to indirectly affecting its phosphorylation state.
Elevated PEVK phosphorylation is associated with increased cardiomyocyte passive stiffness in preclinical models and in human HF (8). To begin to address the therapeutic potential of HDAC6 gain of function, an ex vivo experiment was performed to determine whether HDAC6 is capable of overriding the cardiac stiffening effect of PKC. Myofibrils obtained from nonfailing human LVs were pretreated with recombinant PKCα, with or without the addition of recombinant HDAC6 ( Figure 5B). PKCα dramatically increased myofibril resting tension at physiological sarcomere length (2.0-2.2 μm), and, remarkably, this stiffening was completely normalized upon subsequent exposure of the myofibrils to HDAC6 ( Figure  5C). Recombinant HDAC6 also largely blocked the PKC-induced steeper resting tension-to-sarcomere length curve ( Figure 5D). The presence of recombinant HDAC6 did not reduce PKC-driven phosphorylation of cardiac proteins and, if anything, may have augmented titin phosphorylation ( Figure 5, E and F). These data demonstrate the ability of HDAC6 to neutralize PKC-mediated stiffening of human myofibrils through a mechanism that is independent of phosphorylation, suggesting that HDAC6 gain of function could be therapeutically beneficial in the setting of DD.
HDAC6 deletion alters cardiac myofibrillar protein acetylation. To further address the mechanism(s) by which HDAC6 modulates myofibril stiffness, the acetylation state of sarcomeric proteins was evaluated. Immunoblotting demonstrated that reduced myofibril compliance in HDAC6-KO mice was not associated with changes in titin isoform expression or the global acetylation state of titin (Supplemental Figure 6, A-F). Next, mass spectrometry-based acetylproteomics analysis was performed on LV lysates to address the impact of HDAC6 deletion on site-specific lysine acetylation in the heart. Online liquid chromatography-coupled tandem mass spectrometry was performed with affinity-purified acetylated peptides derived from trypsin-digested total LV protein homogenates and revealed an increase in acetylation of several lysines in titin in HDAC6-KO hearts, including 2 sites near the PEVK element (K13013 and K13597) (Supplemental Figure 7A). Acetylation of K191 of cardiac myosin-binding protein C (MyBP-C) was also increased in HDAC6-KO hearts (Supplemental Figure 7A). Annotated MS2 spectra confirmed the identity of specific lysine acetylation sites in titin and MyBP-C (Supplemental Figure 7, B-D). These findings suggest that HDAC6 deacetylates multiple lysine residues within cardiac sarcomeric proteins, and support a role for HDAC6 as a titin deacetylase.
HDAC6 loss of function exacerbates DD in mouse models. We hypothesized that DD is exacerbated by HDAC6 deletion as a result of elevated passive stiffness of the heart. To test this, HDAC6-KO mice and WT controls were evaluated in a model of hypertension-induced DD with preserved ejection fraction driven by combined uninephrectomy (UNX) and deoxycorticosterone acetate (DOCA) ( Figure 6A and Supplemental Table 1). Serial Doppler echocardiography was used to quantify diastolic function by measuring the ratio of the early filling (E) phase of the LV during diastole, which is due to relaxation of the LV, and the late filling (A) phase, which is mediated by contraction of the atrium. Two and four weeks after UNX/DOCA, HDAC6-KO exhibited more severe DD than WT controls, as evidenced by pronounced reduction in E/A ( Figure 6, B and C). Doppler measurements of septal mitral annulus velocity (E′/A′) confirmed the more rapid onset of DD in HDAC6-KO mice compared with WT controls (Figure 6, D and E). At the study endpoint of 6 weeks, E/A and E′/A′ were equivalent between HDAC6-KO and WT mice. Nonetheless, invasive hemodynamic measurements obtained at this time confirmed that HDAC6-KO mice subjected to UNX/DOCA had elevated LV end-diastolic pressure compared with controls ( Figure 6F), as well as a greater degree of exercise intolerance ( Figure 6G), which correlates with DD (32). WT and HDAC6-KO mice had preserved ejection fraction throughout the 6-week study ( Figure 6H). DD is often attributed to cardiac hypertrophy and fibrosis. However, HDAC6-KO and WT mice developed equivalent hypertrophy in response to UNX/DOCA, as determined by LV mass-to-tibia length measurements and echocardiographic assessment of LV wall thickness ( Figure 6I and Supplemental Table 2). Picrosirius red staining of LV sections failed to reveal significant interstitial fibrosis in any of the groups ( Figure 6J).
To further address the mechanism of more severe DD in HDAC6-KO mice, a repeat study was performed, with analyses focusing on the 2-week time point, when the difference in DD between WT and HDAC6-KO mice was most exaggerated (Figure 6K). Tail cuff measurements revealed that mice subjected to UNX/DOCA remained normotensive 2 weeks after surgery (Figure 6L), demonstrating that the observed DD at this early stage occurred independently of high blood pressure. In contrast, resting tension-to-sarcomere length curves showed that UNX/DOCA treatment for 2 weeks led to stiffening of LV myofibrils, and the reduction in myofibril compliance was exaggerated in mice lacking HDAC6 ( Figure 6M). These data suggest that the intensified DD in HDAC6-KO mice is due to increased stiffening of titin.
Given that aging is an independent risk factor for the development of DD, we also performed serial echocardiography on normotensive WT and HDAC6-KO mice over the course of 18 months. Ejection fraction was preserved in WT and HDAC6-KO J Clin Invest. 2022;132(10):e148333 https://doi.org/10.1172/JCI148333 Prior acetylproteomics studies have shown a number of titin lysine residues to be acetylated in rodent hearts (33,34). Importantly, one of these sites corresponds to mouse K12905 near the PEVK/Ig-like region of titin that is necessary for recombinant HDAC6 to reduce myofibril stiffness. In addition, K12905 and 5 additional lysines in this region were found to be acetylated in skeletal muscle titin (34,35), with one site (K12868 in mouse) showing elevated acetylation in response to Scriptaid (35), a hydroxamic acid HDAC inhibitor with a selectivity profile similar to that of ITF2357. It has been proposed that, upon phosphorylation, introduction of negatively charged phosphate groups to sites in the highly basic PEVK domain increases titin-based passive stiffness by altering ionic intramolecular interactions (8). It is possible that neutralization of the positive charge of lysine by acetylation in response to HDAC6 inhibition increases titin stiffness by further enhancing these intramolecular interactions. Consistent with this notion, using an ex vivo system with isolated myofibrils from human heart, we demonstrated that PKCα-mediated titin stiffening can be reversed by treatment of myofibrils with recombinant HDAC6.
Immunoblotting with anti-acetyl-lysine antibodies confirmed that cardiac titin is acetylated, but we failed to note a significant difference in this global titin acetylation signal between WT and HDAC6-KO hearts, suggesting that HDAC6 selectively deacetylates one or a small subset of the total acetylated lysines on titin. In support of this notion, subsequent mass spectrometry analysis revealed site-specific increases in titin acetylation in HDAC6-KO hearts compared with WT controls. Assessment of the impact of converting these lysines to arginine, which cannot be acetylated, or glutamine, which sometimes functions as an acetyl-lysine mimetic, will be required to assess their potential roles in the regulation of myofibril stiffness. It is also possible that HDAC6 regulates diastolic function by deacetylating sarcomeric proteins other than titin. In this regard, we observed an increase in MyBP-C acetylation in HDAC6-KO hearts compared with WT controls, and mutations in the gene encoding this protein have been shown to trigger DD (36). Implementation of more quantitative proteomics methods, such as stable isotope labeling by amino acids (SILAC), would yield a comprehensive inventory of lysines in titin and other myofibrillar proteins that are substrates for HDAC6-mediated deacetylation.
It is not known whether our findings are related to the recent demonstration that increasing NAD + by administering its precursor, nicotinamide, ameliorates DD in association with reductions in titin stiffness and acetylation state in murine models (19). While nicotinamide is predicted to stimulate sirtuin activity, not HDAC6 activity, prior work has established that HDAC6 and sirtuins can function redundantly by deacetylating common substrates (37). Thus, it is possible that HDAC6 and sirtuins modulate myofibril stiffness through similar mechanisms.
A paradoxical finding from the current study is that tubastatin A, which selectively inhibits HDAC6, increases titin stiffness, while ITF2357, which inhibits HDAC6 as well as multiple other zinc-dependent HDACs, fails to alter the passive tension of myofibrils. This is not due to differences in potency or cellular activity of the compounds, since tubastatin A and ITF2357 inhibit HDAC6 with low-nanomolar potency, and both dramatically increased mice throughout the duration of the study (Supplemental Figure  8A). However, consistent with findings from the UNX/DOCA model, HDAC6-KO mice exhibited signs of impaired diastolic function with aging compared with WT controls (Supplemental Figure 8, B and C, and Supplemental Table 3). Evaluation of a greater number of mice, and with more advanced age, will be required to corroborate these findings.

Discussion
By identifying HDAC6 as a regulator of myofibril stiffness, the current study provides evidence to support a role for reversible lysine acetylation in the control of the elastic properties of titin, and titin-based passive stiffness of the myocardium. We initially established a putative connection between HDAC6 and titin by evaluating the mechanical properties of isolated myofibrils from WT and HDAC6-KO mice ex vivo. Deletion of HDAC6 augmented myofibril stiffness in a manner that persisted in the presence of BDM, which disrupts actomyosin cross-bridges, strongly suggesting that HDAC6 modulates myofibril compliance via titin. Conversely, treatment of murine or human myofibrils with recombinant HDAC6 led to enhanced compliance. Importantly, the ability of HDAC6 to alter myofibril elastic properties was lost upon deletion of 282 amino acids of titin encompassing the approximately 190 carboxy-terminal residues of the PEVK element and an adjacent Ig-like domain. Thus, we propose that HDAC6 regulates myofibril compliance, at least in part, through deacetylation of lysines embedded within, or adjacent to, this region of titin, and possibly through deacetylation of other sarcomeric proteins (Figure 7). based ectopic expression of HDAC6 in cardiomyocytes, should be considered as an alternative or complementary approach to enhance titin compliance for the treatment of patients with DD. Conversely, DCM with systolic dysfunction is associated with increased myofilament compliance and submaximal force generation, leading to speculation that titin-stiffening agents could enhance contractility and have therapeutic benefit in the context of HF with reduced ejection fraction (HFrEF) (6). The possible utility of HDAC6 inhibition for the treatment of HFrEF is underscored by the prior demonstration that HDAC6 catalytic activity is elevated in models of HF (43), and HDAC6 KO and/or tubastatin A administration improves systolic function in mouse models of pressure overload, angiotensin II infusion, doxorubicin treatment, and ischemia/reperfusion (22,44,45). Furthermore, HDAC6 inhibition blocks the formation of cardiotoxic protein aggregates associated with desmin-related cardiomyopathy (46). Nonetheless, the extent to which titin alterations contribute to the efficacy observed in these models is unclear.
Evaluation of HDAC function in the heart has historically focused on epigenetic regulation of gene transcription through removal of acetyl groups from histone tails. However, to date, the most prominent functions of HDAC6 have been noted in the cytoplasm, where it controls the acetylation state of cytoskeletal proteins such as tubulin (24). Thus, HDAC6 is a particularly attractive target since its inhibition largely avoids untoward effects of broadbased epigenetic regulation throughout the body (47). Mouse knockout studies highlight this point. Despite having mild DD, as described here, mice with global deletion of HDAC6 develop normally and do not exhibit overt pathological phenotypes (48). In contrast, whole-body deletion of HDACs that serve fundamental roles in the control of histone deacetylation, such as HDAC1 and HDAC2, leads to early embryonic or perinatal mortality (49). Multiple HDAC6-selective inhibitors are in clinical development for indications such as cancer, neurodegeneration, and autoimmune diseases and, to our knowledge, have not elicited adverse effects in humans (20). Nonetheless, our findings suggest that HDAC6-selective inhibition should be avoided in individuals presenting with DD, since myofibril stiffening would be predicted to aggravate the ventricular filling defect. This concept is supported by the demonstration that HDAC6-KO mice developed more severe DD than controls in the UNX/DOCA model, and that HDAC6-KO mice have a propensity to develop exaggerated DD with aging compared with WT controls. Thus, at the level of cardiac myofilament, manipulation of HDAC6 can be both beneficial and detrimental, emphasizing the need to carefully titrate the function of this deacetylase, either positively or negatively, for therapeutic purposes.
Limitations and conclusions. A limitation of our study is that we did not demonstrate that HDAC6 directly deacetylates titin. Future identification of lysine acetylation sites that regulate myofibril stiffness will facilitate assessment of direct versus indirect effects of HDAC6 on titin compliance. Furthermore, although the mouse UNX/DOCA model is useful for studying DD in the context of preserved ejection fraction, the animals do not develop symptoms of overt HF. Thus, additional work is needed to determine whether HDAC6 inhibition exacerbates other hallmark features of HFpEF, such as lung edema. tubulin acetylation in ARVMs. We postulate that concomitant inhibition of numerous HDAC isoforms by ITF2357 neutralizes the myofibril stiffening action of HDAC6 inhibition, perhaps by enhancing acetylation of lysine residues that increase titin compliance. This scenario would be analogous to the mode in which kinases differentially modulate titin compliance based on the position of the target phospho-acceptor sites (8). Nonetheless, we cannot rule out the possibility that the inability of ITF2357 to stiffen myofibrils is governed by an alternative HDAC-dependent and/or off-target mechanism. We previously demonstrated that ITF2357 and the related pan-HDAC inhibitor suberoylanilide hydroxamic acid (SAHA) improve diastolic function in murine and feline models of hypertension, aging, and chronic pressure overload in association with enhanced myofibril relaxation (21,38). The inability of pan-HDAC inhibition to alter titin compliance likely explains why ITF2357 and SAHA failed to exacerbate DD, and instead elicited protective actions in the heart through distinct mechanisms, such as enhancing the acetylation state of troponin I, which augments myofibril relaxation (39). The relative contributions of impaired myofibril relaxation and titin stiffening to the pathogenesis of DD remain to be determined.
It is important to note that changes in the viscoelastic properties of the microtubule network have recently been shown to contribute to cardiac stiffening and DD (40). Since HDAC6 is a tubulin deacetylase, it is conceivable that part of its ability to modulate diastolic function in vivo is mediated via microtubules. Nonetheless, it is improbable that the myofibril preparations used in our ex vivo assays contained intact microtubules, and thus the mechanical changes observed following HDAC6 inhibition or activation are likely due to direct effects on the acetylation state of titin or other sarcomeric proteins.
Therapeutic strategies designed to enhance titin compliance have focused on PKG activation and, more recently, on development of small-molecule RBM20 inhibitors and antisense oligonucleotides to promote expression of larger, more compliant titin isoforms (41,42). Based on our findings, HDAC6 gain of function, either pharmacologically or through gene therapy- HDAC6-mediated deacetylation reduces myofibril stiffness, whereas inhibition of HDAC6-mediated deacetylation leads to myofibril stiffening. We propose that HDAC6 regulates myofibril compliance, at least in part, through deacetylation of lysines embedded within, or adjacent to, the PEVK element of titin, and possibly through deacetylation of other regions of titin and/or other sarcomeric proteins.
Bioscience) for 30 minutes at 15°C. For the treatment of recombinant PKCα and recombinant HDAC6, myofibril lysates were resuspended in 150 μL of relaxing solution containing recombinant PKCα (0.066 U/μL; Millipore 14-484) and lipid activator (Millipore 20-133) for 30 minutes with or without the addition of recombinant HDAC6 (1 μg/ μL; BPS Bioscience) for another 30 minutes. After treatment, myofibril lysates were washed twice with relaxing solution containing protease inhibitors, and mechanics studies were performed. Homogenized myofibrils were centrifuged at 13,000g for 5 minutes, then separated using agarose gels.
Statistics. Mean ± SEM values are shown and were compared by 2-tailed Student's t test (2 unpaired groups), 1-way ANOVA (>2 groups), or mixed-effects models for repeated measures followed by Tukey's or Bonferroni's multiple-comparison tests where appropriate. A P value less than 0.05 was considered statistically significant.
Study approval. Animal experiments were reviewed and approved by the Institutional Animal Care and Use Committee at the University of Colorado Anschutz Medical Campus. Human hearts were obtained from a tissue bank, which was reviewed and approved by the Colorado Multiple Institutional Review Board (COMIRB 01-568) and is maintained by the Division of Cardiology at the University of Colorado Anschutz Medical Campus. Non-failing human hearts were obtained from individuals who provided written informed consent to be donors. In conclusion, our findings suggest a role for HDAC6-reversible acetylation as a posttranslational modification that controls myocardial passive stiffness. The data underscore emerging nongenomic roles for HDACs in the heart, and the potential to develop unique interventions for distinct forms of HF based on the ability to influence the elastic properties of titin by manipulating the catalytic activity of HDAC6.

Methods
Further information can be found in Supplemental Methods.
Myofibril mechanics analyses. Myofibril mechanics were quantified using the fast solution switching technique (21,50). Frozen LV sections were skinned in 0.5% Triton X-100 in rigor solution (132 mM NaCl, 5 mM KCl, 1 mM MgCl 2 , 10 mM Tris, 5 mM EGTA, pH 7.1) containing protease inhibitors (10 μM leupeptin, 5 μM pepstatin, 200 μM PMSF, and 10 μM E64) as well as 500 μM NaN 3 and 500 μM DTT at 4°C overnight. Skinned LVs were washed in fresh rigor solution and homogenized (Tissue-Tearor, Thomas Scientific) in relaxing solution (pCa 9.0, where pCa = -log 10 [calcium concentration]) containing protease inhibitors. Myofibril suspensions were transferred to a temperature-controlled chamber (15°C) containing relaxing solution (pCa 9.0; 100 mM Na 2 EGTA, 1 M potassium propionate, 100 mM Na 2 SO 4 , 1 M MOPS, 1 M MgCl 2 , 6.7 mM ATP, and 1 mM creatine phosphate, pH 7.0). Myofibril bundles were mounted between 2 microtools. One tool was connected to a motor that could produce rapid length changes (Mad City Labs). The second tool was a calibrated cantilevered force probe (10.75 μm/μN; frequency response 2-5 kHz) . Myofibril length was set at approximately 2.2 μm. Average sarcomere lengths and myofibril diameters were measured using ImageJ software (NIH). Mounted myofibrils were activated and relaxed by rapid translation of the interface between 2 flowing streams of solutions of different pCa. Data were collected and analyzed using customized LabView software. Measured mechanical and kinetic parameters were defined as follows: resting tension (mN/ mm 2 ) = myofibril basal tension in fully relaxing condition; maximal tension (mN/mm 2 ) = maximal tension generated at full calcium activation (pCa 4.5); the rate constant of tension development following maximal calcium activation = k ACT ; duration of the linear relaxation = linear duration; and the rate constant of exponential relaxation = fast k REL . For myofibril resting tension-to-sarcomere length curves, each myofibril was stretched incrementally to determine the resting tension at different sarcomere lengths, and a resting tension-sarcomere length relationship was measured. The data from all myofibrils of the same animal were grouped into 0.15-μm intervals of sarcomere length and averaged. The averaged data of all animals from each group were shown as mean ± (or +) SEM values fitted by third-order polynomials. At least 1 sample from each treatment group or genetic background was analyzed on a given day. All cultured cells in a particular experiment were harvested on the same day. While myofibril experiments were conducted by the person preparing the samples and thus were not blinded to genotype or drug treatment, the data were analyzed in a blinded fashion.


s2fieldsofstudy: Biology Medicine

text:
Inefficiency of wide-based walking from the perspective of lateral center of gravity, gluteus medius muscle myoelectric activity, and cardiopulmonary parameters

[Purpose] To clarify the inefficiency of wide-based walking from kinematic and exercise physiology perspectives. [Participants and Methods] Participants consisted of 20 healthy male university students who performed treadmill walking under conditions of normal walking and wide-based walking (20-cm stride width). The lateral center of gravity movement, gluteus medius muscle myoelectric activity, oxygen uptake, minute ventilation (tidal volume, respiratory rate), heart rate, blood pressure, and rating perceived exertion just before the end of constant load exercise (4.0 km/h) were compared between the two walking conditions. [Results] All the measured parameters except for tidal volume and diastolic blood pressure were significantly higher during wide-based walking than during normal walking. However, when Δ is the difference between the two conditions, no correlation was found between Δlateral center of gravity movement, Δgluteus medius muscle myoelectric activity, Δcardiopulmonary parameters, and Δrating perceived exertion. [Conclusion] Although the precise mechanisms underlying the inefficiency of wide-based walking could not be clarified, cardiopulmonary indices such as oxygen uptake were significantly higher during wide-based walking than during normal walking. This suggests that improvement of wide-based gait is warranted from a kinematic perspective and an exercise physiology perspective.

INTRODUCTION
A wide-based gait with increased stride width is a common feature among patients with balance disorders that becomes more prominent as the disease progresses, such as spinocerebellar degeneration. Although this gait is thought to represent a compensatory strategy to increase the base of support in order to walk without falling, attempts are still made to bring the gait closer to normal through physical therapy. One reason for such attempts is to improve gait appearance, and another is to increase the efficiency of walking. A previous study by Kawahara et al. 1) found that displacement of the center of gravity (COG) of the body was significantly increased in the left-right direction when walking with an enlarged stride width, such as wide-based walking, and that maximum knee joint valgus moment in the early to mid-stance phase decreased, hip joint adduction moment decreased, and ankle joint varus moment increased as stride width increased. However, studies have yet to confirm whether wide-based walking is inefficient compared to normal walking, which is considered the most efficient form of walking.
The purpose of this study was therefore to clarify the inefficiency of wide-based walking from the perspectives of lateral COG movement, gluteus medius muscle myoelectric activity, and cardiopulmonary parameters.
Participants were 20 healthy male university students. Mean ± standard deviation (SD) of age of the participants was 21.9 ± 1.2 years, mean height was 174.7 ± 4.9 cm, mean weight was 66.9 ± 10.1 kg, and mean body mass index (BMI) was 21.9 ± 2.5 kg/m 2 . The purpose, methods, and freedom to take part in or withdraw from the study were explained to all participants, and written consent for participation was obtained prior to enrolment.
Participants were made to walk on a treadmill (MAT-3200; Fukuda Denshi, Tokyo, Japan) under conditions of normal walking and wide-based walking (20-cm stride width). The order of conditions was randomized, and participants were instructed to walk while stepping on the treadmill with heel contact first. Participants were asked to perform the different conditions at intervals of at least 24 h. Walking speed on the treadmill was kept at a constant 4.0 km/h, and participants walked for 5 min after a 3-min warm-up. The following items were measured during walking: lateral COG movement; gluteus medius muscle myoelectric activity; cardiopulmonary parameters; and rating perceived exertion (RPE).
A Gait-kun triaxial acceleration gait analyzer (MG-M1110; LSI Medience, Tokyo, Japan) was used to measure COG movement during walking. The Gait-kun was attached to the waist via an accessory belt, and mean lateral movement was measured. Data obtained from the Gait-kun were analyzed using Gaitview MG-M1110 software (LSI Medience). The value of lateral COG movement was obtained from 10 steps just before the end of walking.
Surface electromyography (EMG) was performed on the right gluteus medius muscle using an Ultium EMG system (NORAXON EM-U810M8; SakaiMed, Tokyo, Japan). Electrodes were attached parallel to the muscle fibers with a center distance of 20 mm. The electrode was attached halfway along the line connecting the iliac crest to the greater trochanter, in accordance with the Surface ElectroMyoGraphy for the Non-Invasive Assessment of Muscles (SENIAM) guideline 2) . Before attaching the electrode, the skin was treated with an external skin disinfectant and a skin preparation gel for biological signal monitoring (Skinpure; Nihon Kohden, Tokyo, Japan). Measurement was performed with the participant lying on their left side and raising their right leg. The measurement time was 5 s. After excluding the first and last 1 s, the middle 3 s were analyzed. The root mean square (RMS) of the waveform during maximum voluntary contraction (MVC) was set at 100%, and RMS of the EMG waveform at 4-5 min during walking was displayed as %MVC.
An exhaled gas analyzer (AE310S; Minato Medical Science, Tokyo, Japan) and a load ECG analyzer (MLX-1000 system; Fukuda Denshi, Tokyo, Japan) were used to measure the following cardiopulmonary parameters: oxygen uptake (VO 2 ), minute ventilation rate (VE), tidal volume (VT), respiratory rate (RR), heart rate (HR), systolic blood pressure (SBP), and diastolic blood pressure (DBP). All of these parameters were measured before the start of walking and before the end (average value for 4.5 to 5.0 min) of the 5-min walk.
For RPE, lower limb fatigue and general fatigue were measured before the start of walking and just before the end of the 5-min walk by asking participants to point to a rating on a modified Borg scale.
After confirming the normality of the distribution of parameters using the Shapiro-Wilk test, the paired t-test was used to compare the two conditions. The Wilcoxon signed-rank test was used to compare two conditions when a normal distribution was not observed. In addition, Pearson's product-rate correlation or Spearman's correlation by rank were used to test for the existence of correlations between differences between conditions of COG movement (Δlateral COG movement distance), differences between conditions of muscle activity (Δgluteus medius muscle myoelectiric activity), and differences between conditions of cardiopulmonary parameters(ΔVO 2 , ΔVE, ΔVT, ΔRR, ΔHR, ΔSBP, and ΔDBP) and differences between conditions of RPE (ΔRPE). SPSS version 21.0 software (IBM Japan, Tokyo, Japan) was used for statistical analyses, and the level of significance for all statistical processing was set at 5%.

RESULTS
Both lateral COG movements and the gluteus medius muscle myoelectric activities were significantly higher in the widebased walking than in the normal walking (Table 1). Furthermore, all but VT and DPB of the cardiopulmonary parameters were also significantly higher in the wide-based walking than in the normal walking (Table 2). On the other hand, two items of RPE were not significantly different between the two walking conditions ( Table 2). The correlation coefficient ρ between the Δlateral COG movement and Δgluteus medius muscle myoelectric activity was 0.250, and there was no correlation. In addition, no correlations were found at each Δlateral COG movement and Δgluteus medius muscle myoelectric activity, and Δcardiopulmonary parameters and ΔRPE (Table 3).

DISCUSSION
The present results showed that lateral COG movement was significantly higher in the wide-based walking than in the normal walking, supporting previous findings by Kawahara et al 1) .
Myoelectric activity in the gluteus medius muscle is significantly increased in the wide-based walking compared to normal walking. Kobayashi et al. 3) reported, when stride width was increased from 10 cm (similar to normal walking) to about 30 cm, myoelectric activity in the gluteus medius muscle was not significantly increased, but when stride width was increased from 30 cm to 40 cm, gluteus medius muscle myoelectric activity was increased. In the present study, however, muscle activity was increased at a stride width of 20 cm compared to that during normal walking, which was not significantly different in the multiple comparison test in the study by Kobayashi et al. Only 6 participants were included in their study, and 4 types of stride widths were set, which may be one of the reasons for the difference in the results from our study. Since the stride width during normal walking is 8.0 cm 4) , it is considered that the setting of 20 cm for wide-based walking was sufficient to induce kinematic and exercise-physiological differences. In terms of cardiopulmonary parameters, significant differences were identified in VO 2 , VE, RR, HR, and SBP before the end of exercise. Wide-based walking has higher oxygen consumption, ventilation demand, and cardiovascular response than normal walking, and it has become clear that it is inefficient for walking with the same external exercise load.
We think that the lack of significant difference between the two conditions in terms of RPE, both for the lower limbs and the whole body, was attributable to walking speed. Participants in the present study walked for 5 min at a normal walking speed (level ground, 67 m/min), which is considered to be 3.0 METs, based on the METs table for daily activities in the Physical Activity Standards for Health Promotion 2013 5) . Since this represented moderate-intensity activity 6) and the exercise was performed at 3.0 METs (at the lower end of the moderate-intensity range), this exercise was expected to be completed before the participant felt a high level of fatigue in the lower limbs or whole body during either normal or wide-based walking.
No correlation was apparent between Δlateral COG movement and Δgluteus medius muscle myoelectric activity. In addition, no correlation was seen between Δlateral COG movement and Δgluteus medius muscle myoelectric activity, and Δcardiopulmonary parameters and ΔRPE. These results suggest that lateral COG movement and gluteus medius muscle myoelectric activity are not the sole contributors to changes in cardiopulmonary parameters under the two conditions. Our prediction is that wide-based walking increases lateral COG movement compared to normal walking, and increased activity of the gluteus medius muscle suppresses it, resulting in a greater response to cardiopulmonary parameters. However, the suggestion of this study contradicted our prediction. McAndrew Young et al. 7) reported that wide-based walking not only significantly increased stride width, but also significantly shortened step length compared to normal walking. Ohmichi 8) also stated that HR and VO 2 were the lowest in spontaneous walking at any speed, and that physiological intensity increased with both longer and shorter step lengths. Since only stride width was specified in our study, the possibility remains that the step length affects cardiopulmonary indices thus cannot be ruled out. It is considered that the step length during walking at the same speed affects the difference in the cardiopulmonary response. Kobayashi et al. 3) reported that myoelectric activity in the adductor longus muscle increased significantly while walking with a wider stride width of 40 cm, compared to widths of 20 cm and 30 cm, during the 70%-80% of the stance phase of the gait. This suggests that activity of muscles other than the midriff muscles during wide-based walking may affect cardiopulmonary parameters and RPE. Muscle activity of the tibialis posterior and trunk ventral muscles, in addition to the mid-diastasis and adductor muscles, is also expected to increase from the early to mid-to-late stance. However, since this remains a matter of speculation, the number of test muscles in experiments needs to be increased, and this is regarded as an issue for future investigation.
This study found that lateral COG movement and myoelectric activity of the gluteus medius muscle were not the sole factors explaining changes in cardiopulmonary parameters and RPE during wide-based walking. However, values of cardiopulmonary parameters such as VO 2 were higher during wide-based walking than during normal walking. Wide-based walking is considered an inefficient gait. This suggests the desirability of improving gait for individuals showing wide-based walking, not only from a kinematic perspective, but also from an exercise physiological perspective.

Conflict of interest
The authors declare that there is no conflict of interest regarding the publication of this article.